CN110915819B - Method for extracting isaria fumosorosea toxin and application thereof - Google Patents

Abstract

The invention discloses Isaria fumosorosea (A. fumosorosea)Isaria fumosorosea) The extraction method of the strain SP535 toxin comprises the following steps: (1) adding 0.8-1.2 times volume of organic solvent ethyl acetate into fermentation liquor of Isaria fumosorosea SP535, (2) breaking cell wall by ultrasonic oscillation to accelerate toxin extraction, preferably ultrasonic oscillation for 30-50 min; (3) placing the mixture into a refrigerator at 0-6 ℃ for standing after shaking, separating the extract liquor by using a separating funnel, and reserving the extracted organic phase; (4) and carrying out rotary evaporation on the extracted organic phase to obtain the crude toxin. The invention provides an effective method for extracting isaria fumosorosea toxin through a reasonable experimental scheme, the obtained crude toxin has strong pathogenicity to bemisia tabaci, and the crude toxin can be further developed into biological pesticide for preventing and treating bemisia tabaci or matched with other medicines for use.

Description

Method for extracting isaria fumosorosea toxin and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to the field of biological control, and particularly relates to a method for extracting and identifying isaria fumosorosea toxin.

Background

Some pests have strong resistance to chemicals due to long-term use of chemical pesticides, and many natural enemies of pests are mistakenly killed during control (mengling. measures to protect natural enemies of pests during chemical control of pests [ J ]. north river agriculture, 2012, (03): 35-37). With the rapid development of modern green food production, the traditional pest control mode needs to be changed, and a sustainable green treatment way needs to be searched urgently (Qi Liang Bengchen. comprehensive development and research of entomopathogenic fungi [ A ] the mycology society of Chinese plant society, China entomogenous fungi research and application (first volume) [ C ] the mycology society of Chinese plant society, 1986: 10). At present, the proportion of biological control in the disease and insect pest control in China is only 5%, the difference from the actual production requirement is large, and in addition, pollution-free vegetables, fruits and green foods are favored in the market, which means that the biological control has wide application prospect (helium, well-known, Zhonghui, etc.. drug resistance gene mutation screens high-yield strains [ J ]. Jiangxi chemical industry, 2004, (04): 169-. In particular, bemisia tabaci is widely spread and outbreaks of disasters around the world, is a polyphagic pest, numerous in biotypes, wide in hosts and extremely harmful. Bemisia tabaci produces direct damage by sucking sap of host plants, and also produces indirect damage by secreting honeydew to induce sooty mould and spread virus diseases, thus causing great loss to agricultural production in many countries. Bemisia tabaci spreads over 150 virus species, of which tomato yellow leaf curl disease caused by geminivirus is a devastating disease that can lead to 100% loss of tomatoes in severe cases and has spread to other crops. Due to abuse of chemical insecticides, bemisia tabaci has developed resistance to a variety of insecticides. Isaria fumosorosea plays an important natural control role on the growth and the disappearance of bemisia tabaci populations in nature, and is an important pathogenic microorganism of bemisia tabaci.

Entomogenous fungi are abundant in resources, and more than 800 species (Yangyuan, Dukai Books, Shimingwang. biological control research progress of entomogenous fungi [ J ]. proceedings of Henan academy of science and technology (Nature edition), 2011,39(01):34-37) are recorded, and mainly belong to Ascomycetes and zygomycota, wherein Beauveria bassiana (Beauveria bassiana) and Metarrhizium anisopliae (Metarhizium) of the Ascomycetes are developed into important pest biocontrol agents. The pathogenic fungi has the advantages of low toxicity, no pollution, strong sustainability, difficult generation of resistance by pests and the like, and can effectively avoid a series of problems of pesticide residue standard exceeding, pest drug resistance increasing and the like caused by chemical pesticides. The development of the insecticidal fungus pesticide can ensure that chemical control and biological control are fully coordinated and used, so that the pest control work is more reasonable and effective. The method for controlling pests by using entomogenous fungi is mainly characterized by applying beauveria bassiana to control pests such as soybean pod borer and the like (Wanfanghao, Yezhengchu, Guojiangying, and the like, progress and prospect of biological control research in China [ J ] insect knowledge, 2000, (02): 65-74). Therefore, the development and research of efficient and stable-quality insecticidal fungal pesticides have become a key problem for pest control.

In recent years, with the annual spread and the aggravation of small insects such as aphids and whiteflies, the control effect of isaria fumosorosea on whiteflies and aphids has received much attention from researchers in various countries. In the late 80 s of the last century, spores produced by PF97 microcapsule by university of California and W.R.Grace in the last century were sprayed 6 times and 10 times during the growth period of orangutan (about 100 days)⁷spores/mL suspension, significant control was achieved. Thereafter Blicon and Biobest in the Netherlands, Pae-sin in Mexico and Bemisin in Venezuela and Pre, a mixed preparation of blastospores and mycelia developed in the United states and EuropeSequential publication of FeRal (Zhang Wei, Lvli, what's complement, etc. Association of Isaria fumosorosea with short-lived 35 ℃ high temperatures on 3 rd larvae of diamondback moth [ J]Environmental insect bulletin, 2013,35(02): 182-. Although there are many advantages to the use of Isaria fumosorosea for pest control, due to the disadvantages of long insecticidal cycle, large influence by environmental factors, short storage period, unstable control effect and the like in the application process, in order to fully utilize the potential of Isaria fumosorosea, the researches on the biological characteristics of the Isaria fumosorosea and the mixed use of the Isaria fumosorosea and the chemical pesticide are more and more, mainly the researches on the aspects of formulation, mixing property, compatibility, durability and the like (yellow shock, conception, Wujiahui. Paecilomyces fumosorosea and imidacloprid have combined control effect on Bemisia tabaci population [ J]Ecological news 2006,26(10): 3250-.

In recent years, secondary metabolites of entomogenous fungi have attracted great interest (Song river Jing et al, 2011), and after a lot of fungi infect insects, not only grow in the body of the insects in a large amount, and deprive the insects of nutrition, but also toxic secondary metabolites are often produced, so that the insects are diseased and die, and the secondary metabolites are called toxins (LiZengzhi. Typha hibernalong. entomopathogenic mycology [ M ]. Anhui scientific and technological publishers, 1996). Entomogenous fungi form different kinds of toxins, and some facultative pathogenic fungi and even saprophytic fungi can also produce secondary metabolites that are toxic to insects (xiaoling, xiaoxing. biodiversity and forest pest control management and control [ J ]. chinese horticultural abstracts, 2012,28(11): 191-192). Early in the discovery of entomopathogenic mycotoxins, there was an assumption that mycotoxins could be developed into commercial insecticides, and current findings on entomopathogenic fungi have been mainly obtained by studying toxins produced in vitro by fungi (Liu Sha Hua. Paecilomyces fumosoroseus insecticidal toxin isolation and the effect on several metabolic enzymes of aphids [ D ]. Shanxi university of agriculture, 2005).

The secondary metabolites produced by isaria fumosorosea have insecticidal activity, can degrade the components of body walls such as protein, chitin and lipid, and play a key role in the process of killing pests (extraction, separation and structural identification of Ruyanyang. metarhizium anisopliae crude toxin and toxicity determination on several important pests [ D ]. Yangzhou university, 2007), penicillin separated from isaria fumosorosea such as Osborne (1994) can rapidly kill hosts. Beauveria bassiana lactide is obtained from an extract of isaria fumosorosea hyphae by an enzyme-linked immunosorbent mass spectrometry method, different strains are found to be slightly different in toxin content and composition conformation, the compound is separated by reverse high performance liquid chromatography, and the structure of the compound is mainly beauveria bassiana lactide L (about 60 percent) and beauveria bassiana lactide La (about 40 percent) through analysis (Milner, 1997). According to mass spectrum and nuclear magnetic resonance analysis, the structures of the beauveria bassiana lactide L and the beauveria bassiana lactide La are similar to that of beauveria bassiana lactide II reported by Fargues (1996) (Liangli, Tianjing, Manrui Swallow. Isaria fumosorosea research progress [ J ]. Shanxi university of agriculture bulletin (Nature science edition), 2013,33(04): 362-.

Disclosure of Invention

The strain SP535 is separated from soil beside the old Junshan road in Shen agriculture and forestry areas in Hubei province, is a Chinese indigenous strain, has good biocontrol potential and has good control effect on Bemisia tabaci SP 535. Further, the inventors found that Isaria fumosorosea SP535 has a good biodegradation effect on PCA contamination. The Isaria fumosorosea strain is classified and named Cordyceps fumosorosea, is preserved in Guangdong province microorganism culture collection center (GDMCC) in 2018, 12 and 10, and has the preservation number of GDMCC NO:60514 and the preservation address of Guangzhou China (see the prior application No. 201910449530.7 of the inventor, the invention name: the application of Isaria fumosorosea SP535 as entomogenous fungus in p-chloroaniline biodegradation).

The extraction, separation and purification and structural identification of entomogenous mycotoxin are important parts of the infection pathogenic mechanism and toxin generation action mechanism of the insect of the mycotoxin. Therefore, the invention carries out high-toxicity strain screening by taking isaria fumosorosea SP535 and bemisia tabaci as materials, compares the toxin yield and the pathogenic effect of different extraction methods, and finally obtains the best method for extracting toxin, aims to lay a foundation for the development and utilization of isaria fumosorosea insecticidal toxin and provide support for further application research of novel microbial pesticides.

Thus, the present invention provides a method for extracting isaria fumosorosea toxin from isaria fumosorosea SP535, comprising the steps of:

(1) adding 0.8-1.2 times volume, preferably 1 time volume of organic solvent ethyl acetate into fermentation liquor of Isaria fumosorosea SP 535;

(2) ultrasonic oscillation to break cell wall and accelerate toxin extraction, preferably ultrasonic oscillation for 30-50min, most preferably 40 min;

(3) after shaking, the mixture is placed in a refrigerator at 0-6 ℃ for standing, the extract liquid is separated by a separating funnel, and the extracted organic phase is reserved. Preferably, the mixture is placed into a 4-refrigerator for standing for 20-28h, and is stirred once every 7-9h during the standing;

(4) evaporating the extracted organic phase to obtain crude toxin, preferably by rotary evaporation.

Further, the protein concentration is determined in the steps (1), (3) and/or (4), and the toxin production and extraction effect are monitored. The reason for this is that the isaria fumosorosea hyphae are accompanied by the production of protein during the production of toxin by fermentation, and the measurement of the total protein concentration of the fermentation broth is often used for efficient monitoring of toxin production.

Wherein the fermentation liquor of Isaria fumosorosea SP535 is prepared by the following method:

carrying out shake flask fermentation on isaria fumosorosea spore suspension in a constant temperature shaking table, carrying out suction filtration on a bacterial liquid to obtain hypha and fermentation liquor, and preferably centrifuging the fermentation liquor for later use. Wherein, more preferably, the centrifugation is used after the fermentation broth is briefly centrifuged at 8000 rpm/min.

The invention also provides the isaria fumosorosea crude toxin obtained by the method. Which contains the following toxins:

the toxin extracted by the invention has strong pathogenicity on the bemisia tabaci, and can be further developed into biological pesticide for preventing and treating the bemisia tabaci or matched with other medicines for use. Therefore, the invention also provides application of the crude toxin prepared by the method in controlling bemisia tabaci. In one embodiment, the prepared attenuated toxin can be prepared into liquid dosage forms with different concentrations. The application is to spray the crude toxin liquid on crops to prevent and treat bemisia tabaci.

The effects of the present invention are shown in the following aspects: the method comprises the steps of screening various organic solvents to extract toxins in Isaria fumosorosea fermentation liquor, measuring the concentration of the obtained organic phase protein, obtaining ethyl acetate with the best effect of organic solvent extraction, further carrying out rotary evaporation on the organic phase to obtain crude toxins, and measuring the concentration of the crude toxin protein, wherein the best purification effect after organic solvent extraction is ethyl acetate, and the toxins extracted by the ethyl acetate are relatively more and are up to 20 toxins. Furthermore, the ethyl acetate extraction of crude toxin was most pathogenic to Bemisia tabaci, and the corrected mortality rate of Bemisia tabaci was 85.56% at 7 days after infestation, which was much higher than that of Isaria fumosorosea SP535 spore suspension (1.0X 10)⁷spore/mL), fermentation broth after shake flask fermentation corrected mortality seven days after treatment (64.44%, 63.33%, respectively).

In addition, the invention also explores a high performance liquid chromatography-mass spectrometry (LC-MS) sample preparation method and test parameters, and finally determines the optimal parameters. The method comprises the steps of dissolving the extracted crude toxin by using the same reagent, placing the dissolved crude toxin in a fume hood, after an organic solvent is evaporated, dissolving the dissolved crude toxin by using chromatographic pure isopropanol, and testing under the following test conditions: mobile phase a acetonitrile (0.1% formic acid), B contains 0.1% formic acid +10mM aqueous ammonium formate solution, flow rate: 100. mu.l/min, sample size: 5 μ l, detection wavelength: 215 nm. The preferred method of the invention yields isaria fumosorosea toxin which is then dissolved in ethyl acetate for the subsequent test. The test method has the advantages that different types of toxins can be separated as much as possible, the aim of accurate quantitative detection is fulfilled, and the detection accuracy is greatly improved.

Drawings

FIG. 1 Standard Curve of protein concentration

FIG. 2 Strain SP535 variation in protein concentration with different treatments

In the figure, the concentrations of different treatment proteins of strain SP535 are shown. 1. The concentration of the protein in the fermentation liquid is 2, the concentration of the protein in the organic phase after ethyl acetate extraction is 3, the concentration of the protein in the organic phase after methanol extraction is 4, the concentration of the protein in the organic phase after n-hexane extraction is 5, the concentration of the crude toxin protein after ethyl acetate extraction is 5, the concentration of the crude toxin protein after methanol extraction is 6, and the concentration of the crude toxin protein after n-hexane extraction is 7. The data in the figure are the mean and standard error of 3 replicates. Data are followed by the same letter, indicating no significant difference at the 0.05 level (TUKEY' S method).

FIG. 3 LC-MS identification map of strain SP535 fermentation liquor

FIG. 4 identification of crude toxin LC-MS by extraction of strain SP535 with ethyl acetate

FIG. 5 Strain SP535 methanol extraction crude toxin LC-MS identification map

FIG. 6 strain SP535 n-hexane extraction crude toxin LC-MS identification map

FIG. 7 corrected mortality of two-instar nymphs of Bemisia tabaci treated differently by Strain SP535

Detailed Description

The following description is given by way of development and specific embodiments of the present invention, and is not intended to limit the invention thereto.

The first embodiment is as follows: comparison of protein concentrations extracted by different organic solvents

1. Sterilization of test strains, culture media and utensils

Isaria fumosorosea SP535 is separated from soil beside the road of the old Junshan of the Shennongjian forest region in Hubei province, and is preserved in Guangdong province microorganism culture Collection (GDMCC) 12 and 10 days in 2018, wherein the preservation number is GDMCC NO:60514, and the preservation address is Guangzhou in China.

Potato agar medium (PDA): 200g of potato, 20g of glucose, 15-20g of agar and purified water till 1000 mL. Sterilizing at 121 deg.C for 15 min. The culture medium is used for pure culture of strain plates and inclined planes.

Fermentation medium (Asaff et al, 2005): 30g glucose, 3g yeast extract, 0.39g KH₂PO₄，1.42gNa₂HPO₄·12H₂O，0.60g MgSO4·7H₂O，0.70g NH₄NO₃1.00g KCl, and purified water to 1000 mL. Each 150mL of the fermentation medium was filled in a 500mL conical flask and sterilized at 121 ℃ for 15min for use. The culture mediumIs used for shake flask fermentation.

2. Method for preparing spore suspension

Inoculating a test strain on a PDA (personal digital Assistant) plate, culturing for 7 days in a biochemical incubator with constant temperature of 26 ℃ and illumination (12L:12D), after a large amount of spores are produced, scraping hypha and spores of the fungi by using an inoculating needle into a conical flask filled with 20 mL0.05% Tween-80 sterile water, stirring for 30min by using a magnetic stirrer, after the spores are completely dispersed, filtering bacterial liquid by using double-layer gauze, and discarding undispersed hypha and culture medium residues. Counting with a hemocytometer, adjusted to 1X 10 with 0.05% Tween-80 sterile water⁷spores/mL of spore suspension.

Determination of protein concentration by BCA method

Drawing a standard curve:

(1) 0.5mg/mL of the protein standard was added to a standard well of a 96-well plate in an amount of 0. mu.l, 1. mu.l, 2. mu.l, 4. mu.l, 8. mu.l, 12. mu.l, 16. mu.l, or 20. mu.l, and a standard dilution was added to make up to 20. mu.l, corresponding to standard concentrations of 0mg/mL, 0.025mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, or 0.5mg/mL, respectively.

(2) Add 200. mu.l BCA working solution to each well and leave at 37 ℃ for 20-30min (room temperature for 2 h).

(3) The absorbance at A595 nm was measured using a microplate reader.

(4) The regression equation was developed from the standard curve, see FIG. 1, and the protein concentration of the sample was calculated.

4. Test process for measuring protein concentration of strain SP535 fermentation liquor

(1) Preparing spore suspension of Isaria fumosorosea, adjusting to 1 × 10⁷spores/mL.

(2) Adding 6ml of 1.0X 10 into each bottle of fermentation medium⁷spores/mL spore suspension, each treatment was repeated in triplicate, and the shake flask was allowed to ferment for six days in a constant temperature shaker at 26 ℃ and 180 rpm/min.

(3) And after six days, carrying out suction filtration on the bacterial liquid to obtain hypha and fermentation liquor.

(4) Collecting fermentation liquor, taking 1mL of the fermentation liquor, performing short centrifugation at 8000rpm/min, measuring a light absorption value by using a BCA protein concentration kit, and calculating the protein concentration according to a formula.

5. Extraction of toxin from Isaria fumosorosea SP535 fermentation liquor

The toxin was extracted as follows:

(1) the fermentation liquid obtained in the first example was put into a 150mL clean conical flask, and three organic solvents (ethyl acetate, methanol, n-hexane) having the same volume were added, respectively.

(2) Ultrasonic oscillation is carried out for 40min to break cell wall and accelerate toxin extraction.

(3) After shaking, the mixture was placed in a refrigerator at 4 ℃ and allowed to stand for 24 hours (while stirring once every 8 hours), the extract was subjected to liquid separation with a separatory funnel, the organic phase after extraction was retained, and the protein concentration was measured.

(4) And (4) carrying out rotary evaporation on the extracted organic phase to obtain crude toxin, and determining the protein concentration.

The experimental results are as follows:

the fermentation broth after shake flask fermentation of strain SP535, the organic phase after ethyl acetate extraction, the organic phase after methanol extraction, the organic phase after n-hexane extraction, the crude toxin after ethyl acetate extraction, the crude toxin after methanol extraction, and the crude toxin after n-hexane extraction are compared, as shown in FIG. 2, the results show that: after the ethyl acetate and the methanol are extracted, the protein concentration is increased, the crude toxin protein concentration is obviously increased, and the extraction effect of the ethyl acetate is slightly better than that of the methanol; the protein content is not increased after the normal hexane is used for extracting the fermentation liquor, which indicates that the toxin extraction effect is not ideal.

Three organic solvents are used for extracting toxin in Isaria fumosorosea fermentation liquor, and the concentration of the obtained organic phase protein is measured, so that the organic solvent extraction effect is that ethyl acetate > methanol > n-hexane. And (3) carrying out rotary evaporation on the organic phase to obtain crude toxin, measuring the concentration of crude toxin protein, and extracting the obtained organic solvent to obtain the purification effect of ethyl acetate, methanol and n-hexane.

Example two: characterization and identification of isaria fumosorosea crude toxin

1. High performance liquid chromatography-mass spectrometry (LC-MS) sample preparation method and test parameters

(1) Filtering the fermentation liquor after shaking flask fermentation with a filter membrane, and storing in a refrigerator at 4 deg.C.

(2) Dissolving the extracted crude toxin with the same reagent, placing in a fume hood, dissolving with 1mL of chromatographic pure isopropanol after the organic solvent is evaporated, and storing in a refrigerator at 4 ℃ for testing.

(3) And (3) testing conditions are as follows: mobile phase a acetonitrile (0.1% formic acid), B contains 0.1% formic acid +10mM aqueous ammonium formate solution, flow rate: 100. mu.l/min, sample size: 5 μ l, detection wavelength: 215 nm.

The experimental results are as follows:

1. LC-MS identification result of strain SP535 fermentation liquor

The LC-MS analysis results of the strain SP535 fermentation liquid are shown in FIG. 3 and Table 1. SP535 fermentation liquor contains more components, and 43 small molecular compounds are screened out by comparing with a fungal compound database.

TABLE 1 SP535 fermentation broth major component

2. LC-MS identification result of crude toxin extracted by strain SP535 ethyl acetate

The results of the LC-MS analysis of the crude toxin extracted with ethyl acetate by strain SP535 are shown in fig. 4 and table 2. SP535 ethyl acetate extracts crude toxin and screens 20 kinds of small molecule compounds by comparing with a fungal compound database.

TABLE 2 extraction of the major component of the crude toxin by SP535 ethyl acetate

3. LC-MS identification result of crude toxin extracted by strain SP535 methanol

The results of the LC-MS analysis of the crude toxin extracted with methanol by strain SP535 are shown in FIG. 5 and Table 3. And (3) comparing components in the SP535 methanol extracted crude toxin with a fungal compound database to screen out 18 small molecular compounds.

TABLE 3 SP535 methanol extraction of the major component of the crude toxin

4. Bacterial strain SP535 n-hexane extraction crude toxin LC-MS identification result

The results of LC-MS analysis of crude toxin extracted by normal hexane from strain SP535 are shown in FIG. 6 and Table 4. SP535 n-hexane extracts more components in the crude toxin, and 26 small molecular compounds are screened out by comparing with a fungal compound database.

TABLE 4 SP535 n-hexane extraction of the major component of the crude toxin

5. Toxin contained in metabolite of strain SP535

Common toxins in strain SP535 are: hydrolyzed fumonisins (B3 HFB 3/hydrolyzed Fumonisin B3), Gibberellic acid (Gibberellic acid), ustiloxin A (Ustiloxin A), Aphidicolin (Aphidicolin), Pseurotin D, antibiotics (K252a), Malformin A2, 2-Amino-14, 16-dimethylctadecan-3-ol Ergocristine, Auraserone C, pyranonigrinin A, 15-hydroxymycin, Agistin D.

According to the identification results, the three organic solvents have extraction effect on Isaria fumosorosea, and most toxins are extracted by methanol and ethyl acetate. The fungi contain toxins such as Beauvericin (Beauvericin), Penicillin G (Penicillin G), hydrolyzed Fumonisin B3(B3 HFB 3/hydrolyzed Fumonisin B3), Fumonisin B (Fumitremorginin B) and the like.

Example three: insecticidal activity of Isaria fumosorosea and crude toxin on Bemisia tabaci

And (3) determining the lethal effects of isaria fumosorosea spore suspension, fermentation liquor obtained after shake flask fermentation and crude toxin on bemisia tabaci.

(1) Selecting a plurality of insect-free cotton seedlings with consistent growth vigor and more tender leaves, and sleeving an insect-receiving box;

(2) 150 pairs of bemisia tabaci adults are inoculated into each leaf, placed at the room temperature of 26 ℃, laid for 24 hours and blown out;

(3) after the bemisia tabaci grows to the second age, defining an area by taking 150 nymphs as boundaries, soaking the bemisia tabaci into a treatment solution, taking out the bemisia tabaci after 10 seconds, naturally air-drying the water on the surface of the leaf, and flatly paving the leaf with the front side downward in a culture dish containing agar;

(4)1.0×10⁷the suspension with the spore/mL concentration and the fermentation liquid after shake flask fermentation are soaked in 0.05 percent Tween-80 solution to serve as a blank control. Comparing the crude toxin extracted by different organic solvents with corresponding organic solvents, repeating the steps for three times every time, and observing the crude toxin at the room temperature of 26 ℃;

(5) and (4) checking and recording the death condition of the bemisia tabaci every day, picking out dead insects, performing moisturizing culture, and observing whether pathogenic bacteria grow out.

The experimental results are as follows:

strain SP535 spore suspension (1.0X 10)⁷spore/mL), fermentation liquor obtained after shake flask fermentation, ethyl acetate-extracted crude toxin, methanol-extracted crude toxin and n-hexane-extracted crude toxin are used for treating Bemisia tabaci, the second-instar nymphs of Bemisia tabaci all die, and the cumulative death rates of the nymphs at different times are shown in FIG. 7.

The results show that there are great differences in the rate of disease development of Bemisia tabaci SP535 by the different treatments of Isaria fumosorosea, and that the death of Bemisia tabaci was caused by part of the treatments since day 1 after the treatments. The ethyl acetate extraction of the crude toxin was most virulent to Bemisia tabaci, and the corrected mortality rate of Bemisia tabaci was 85 on day 7 after infestation.56 percent. Isaria fumosorosea SP535 spore suspension (1.0X 10)⁷spore/mL), fermentation broth after shake flask fermentation and methanol extracted crude toxin corrected mortality on day seven post treatment to 64.44%, 63.33% and 72.22%, respectively. In addition, n-hexane extraction of crude toxin was the least pathogenic, with a mortality rate of 26.66% corrected 7 days after infestation, and no increase in mortality rate thereafter.

Claims (9)

1. A method for extracting isaria fumosorosea toxin from isaria fumosorosea SP535, which comprises the following steps:

(1) adding 0.8-1.2 times volume of organic solvent ethyl acetate into fermentation liquor of Isaria fumosorosea SP535, wherein the Isaria fumosorosea SP535 fermentation liquor is prepared by carrying out shake-flask fermentation on spore suspension of Isaria fumosorosea in a constant-temperature shaking table, carrying out suction filtration on bacterial liquid to obtain hypha and fermentation liquor, and centrifuging the fermentation liquor for later use;

(2) ultrasonic oscillating for 30-50min to break cell wall and accelerate toxin extraction;

(3) placing the mixture into a refrigerator at 0-6 ℃ after shaking, stirring the mixture once every 7-9h, separating the extract by using a separating funnel, and reserving the extracted organic phase;

(4) evaporating the extracted organic phase to obtain crude toxin;

wherein the collection number of the Isaria fumosorosea SP535 is GDMCC NO: 60514.

2. The method of claim 1, wherein the concentration of the protein is measured at step (1), step (3) or/and step (4) and the production and extraction of the toxin is monitored.

3. The process according to claim 1, wherein the organic solvent ethyl acetate is added in step (1) in an amount equivalent to the volume of the fermentation broth of Isaria fumosorosea SP 535.

4. The method of claim 1, wherein the evaporation in step (4) is rotary evaporation.

5. The method of claim 1, wherein the step (3) is performed by standing in a refrigerator at 4 ℃ for 20-28 hours.

6. A crude toxin of Isaria fumosorosea obtained by the method of any one of claims 1 to 4.

7. The use of a coarse toxin according to claim 6 for controlling bemisia tabaci.

8. The use of claim 7 wherein the crude toxin is sprayed as a liquid formulation on the crop.

9. A method for detecting crude toxin of Isaria fumosorosea, wherein the crude toxin of Isaria fumosorosea obtained by the method according to any one of claims 1 to 5 is dissolved by ethyl acetate, and the test is carried out after the crude toxin is dissolved by chromatographic pure isopropanol after the organic solvent is evaporated in a fume hood, and the test conditions are as follows: mobile phase A: acetonitrile of 0.1% formic acid, B contains 0.1% formic acid +10mM aqueous ammonium formate solution, flow rate: 100. mu.l/min, sample size: 5 μ l, detection wavelength: 215 nm.

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